The applications of heparin in vascular tissue engineering.

Cardiovascular diseases, among all diseases, are taking the most victims worldwide. Coronary artery occlusion, takes responsibility of about 30% of the yearly global deaths in the world (Heart Disease and Stroke Statistics 2017 At-a-Glance, 2017), raising the need for viable substitutes for cardiovascular tissues. Depending on a number of factors, blocked coronary arteries are now being replaced by autografts or stents. Since the autografts, as the gold standard coronary artery replacements, are not available in adequate quality and quantity, the demand for small diameter vascular substitute comparable to native vessels is rapidly growing. Synthetic grafts have been successfully approved for developing vascular replacements but regarding the special conditions in small-caliber vessels, their use is limited to large-diameter vascular tissue engineering. The major problems associated with the vascular tissue engineered grafts are thrombosis and intimal hyperplasia. Heparin, a negatively charged natural polysaccharide has been used in fabricating vascular grafts since it prevents protein fouling on the surfaces and most importantly, impeding thrombosis. Herein, we focused on heparin, as a multifunctional bioactive molecule that not only serves as an anticoagulant with frequent clinical use but also acts as an anti-inflammatory and angiogenic regulatory substance. We summarized heparin incorporation into stents and grafts and their applicability to restrain restenosis. Also, the applications of heparinzation of biomaterials and heparin mimetic polymers and different approaches invoked to improve heparin bioactivity have been reviewed. We summarized the methods of adding heparin to matrices as they were explained in the literature. We reviewed how heparin influences the biocompatibility of the scaffolds and discussed new advances about using heparin in small-diameter vascular tissue engineering.

Combination of low intensity electromagnetic field with chondrogenic agent induces chondrogenesis in mesenchymal stem cells with minimal hypertrophic side effects.

Background: There are different methods to develop in vitro neo-chondral tissues from adipose-derived stem cells (ADSCs). Application of electromagnetic field (EMF) on ADSCs is one of popular approaches, which results in chondrogenesis. If chondrogenic impact of EMF on ADSCs is supposed to be generalized as a protocol in translational medicine field, possible emergence of early or late hypertrophic maturation, mineralization and inflammatory side effects in chondrogenically differentiating ADSCs should be considered.Methods: The advent of chondrogenic and hypertrophic markers by differentiated cells under standard, platelet-rich plasma (PRP)-based or EMF treatments were monitored. Along with monitoring the expressions of chondrogenic markers, inflammatory and hypertrophic markers, VEGF/TNFα secretion, calcium deposition and ALP activity were evaluated.Results: Accordingly, treatment with %5 PRP results in higher GAG production, enhanced SOX9 transcription, lowered TNFα and VEGF secretions compared to other treatments. Although PRP up-regulates miR-146a and miR-199a in early and late stages of chondrogenesis, respectively, application of EMF + PRP down regulates miR-101 and -145 while up-regulates miR-140 and SOX9 expression.Conclusion: Comparing our results with previous reports suggests that presented EMF-ELF in this study with f = 50 Hz, EMF intensity of less than 30 mT, and 5% PRP (v/v), would facilitate chondrogenesis via mesenchymal stem cells with minor inflammation and hypertrophic maturation.Abbreviations: MSCs: mesenchymal stem cells; TGFß: transforming growth factor-beta; PRP: platelet-rich plasma; ELF-EMF: extremely low-frequency electromagnetic fields; GAGs: glycosaminoglycans; ADSCs: adipose-derived stem cells; VEGF: vascular endothelial growth factor; TNFα: tumor necrosis factor alpha; ALP: alkaline phosphatase.

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate.

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.

The osmolyte type affects cartilage associated pathologic marker expression during in vitro mesenchymal stem cell chondrogenesis under hypertonic conditions.

Stem cells' fate during in vitro differentiation is influenced by biophysicochemical cues. Osmotic stress has proved to enhance chondrocyte marker expression, however its potent negative impacts had never been surveyed. We questioned whether specific osmotic conditions, regarding the osmolyte agent, could benefit chondrogenesis while dampening undesired concomitant hypertrophy and inflammatory responses. To examine the potential side effects of hypertonicity, we assessed cell proliferation as well as chondrogenic and hypertrophic marker expression of human Adipose Derived-MSC after a two week induction in chondrogenic media with either NaCl or Sorbitol, as the osmolyte agent to reach a +100 mOsm hypertonic condition. Calcium deposition and TNF-α secretion as markers associated with hypertrophy and inflammation were then assayed. While both hyperosmotic conditions upregulated chondrogenic markers, sorbitol had a nearly three times higher chondro-promotive effect and a lesser hypertrophic effect compared to NaCl. Also, a significantly lesser calcium deposition was observed in sorbitol hypertonic group. NaCl showed an anti-proinflammatory effect while sorbitol had no effect on inflammatory markers. The ossification potential and cartilage associated pathologic markers were affected differentially by the type of the osmolyte. Thus, a vigilant application of the osmotic agent is inevitable in order to avoid or reduce undesired hypertrophic and inflammatory phenotype acquisition by MSC during chondrogenic differentiation. Our findings are a step towards developing a more reliable chondrogenic regimen using external hypertonic cues for MSC chondrogenesis with potential applications in chondral lesions cell therapy.

Electrospun composite PLLA/Oyster shell scaffold enhances proliferation and osteogenic differentiation of stem cells.

In bone tissue engineering, bioceramics are of the most widely used materials for treatment of bone defects clinically. The composites of bioceramic/polymer fibrous scaffolds have been designed and developed to fulfill the mechanical and biological requirements of the damaged tissue. In the present study, oyster shell (OS) as a bioceramic in combination with the biodegradable and biocompatible poly (l-lactide) has been used to prepare a new tissue-engineered composite. The morphology, porosity, water contact angle and mechanical properties of scaffolds were investigated. Mesenchymal stem cells were also cultured on fabricated scaffolds to evaluate their potential to support cell proliferation and osteogenic differentiation. The SEM results indicated that the electrospun scaffolds were nanostructured and the OS were oriented along the fiber axis. The tensile strength and also the increased surface hydrophilicity of scaffolds after plasma treatment were suitable for tissue engineering applications. MTT assay demonstrated that the fabricated scaffolds were capable of supporting stem cell attachment and proliferation. Biomineralization measurements demonstrated the enhanced osteogenic differentiation of stem cells on composite PLLA/OS scaffolds. Taken together, these scaffolds were shown to hold promising potential for the treatment of bone defects in vivo.

Electrical stimulation of somatic human stem cells mediated by composite containing conductive nanofibers for ligament regeneration.

One of the advances in the field of biomedical nanotechnology, is conductive nanofiber fabrication and the discovery of its applications. Biocompatible flexible nanofibers that have a good biocompatibility, mechanical properties and morphology. Poly (3, 4-ethylene dioxythiophene) (PEDOT) is a conductive polymer that has recently been used in medical applications. In this study, the electrospinning technique and vapor phase polymerization combination method with freeze drying was used to produce Silk fibroin/PEDOT/Chitosan nanocomposite scaffold. The aim of our study was to develop a ligament construct of PEDOT/Silk bilayer nanofibrous scaffold, to mimic the aligned collagen fiber bundles and Chitosan sponge coating was done on these fibrous scaffolds, to mimic the glycosaminoglycans of ECM sheath. The developed constructs were characterized. The unrestricted somatic human stem cells (USSC), were cultured on the scaffold. Then, the effect of applying DC electric pulses to cells cultured on polymer was assessed. Cellular function was actively exhibited in scaffold with electrical induction, as evident by the high expression of collagen I, collagen III, decorin, biglycan and aggrecan genes. Novel scaffold plus electrical stimulation shows facilitating cell seeding and promoting cell proliferation, differentiation. This composites can be used in this new field for stem cells differentiation to target tissues.

Neuroregenerative effects of olfactory ensheathing cells transplanted in a multi-layered conductive nanofibrous conduit in peripheral nerve repair in rats.

BACKGROUND: The purpose of this study was to evaluate the efficacy of a multi-layered conductive nanofibrous hollow conduit in combination with olfactory ensheathing cells (OEC) to promote peripheral nerve regeneration. We aimed to harness both the topographical and electrical cues of the aligned conductive nanofibrous single-walled carbon nanotube/ poly (L-lactic acid) (SWCNT/PLLA) scaffolds along with the neurotrophic features of OEC in a nerve tissue engineered approach. RESULTS: We demonstrated that SWCNT/PLLA composite scaffolds support the adhesion, growth, survival and proliferation of OEC. Using microsurgical techniques, the tissue engineered nerve conduits were interposed into an 8 mm gap in sciatic nerve defects in rats. Functional recovery was evaluated using sciatic functional index (SFI) fortnightly after the surgery. Histological analyses including immunohistochemistry for S100 and NF markers along with toluidine blue staining (nerve thickness) and TEM imaging (myelin sheath thickness) of the sections from middle and distal parts of nerve grafts showed an increased regeneration in cell/scaffold group compared with cell-free scaffold and silicone groups. Neural regeneration in cell/scaffold group was very closely similar to autograft group, as deduced from SFI scores and histological assessments. CONCLUSIONS: Our results indicated that the tissue engineered construct made of rolled sheet of SWCNT/PLLA nanofibrous scaffolds and OEC could promote axonal outgrowth and peripheral nerve regeneration suggesting them as a promising alternative in nerve tissue engineering.

Aligning fermentation conditions with non-canonical amino acid addition strategy is essential for Nε-((2-azidoethoxy)carbonyl)-L-lysine uptake and incorporation into the target protein.

Protein engineering with non-canonical amino acids (ncAAs) holds great promises for diverse applications, however, there are still limitations in the implementation of this technology at manufacturing scale. The know-how to efficiently produce ncAA-incorporated proteins in a scalable manner is still very limited. In the present study, we incorporated the ncAA N6-[(2-azidoethoxy)carbonyl]-L-lysine (Azk) into an antigen binding fragment (Fab) in Escherichia coli. We used the orthogonal pyrrolysyl-tRNA synthetase/suppressor tRNACUAPyl pair from Methanosarcina mazei to incorporate Azk site-specifically. We characterized Azk uptake and Fab production at bench-scale under different fermentation conditions, varying timing and mode of Azk addition, Azk-to-cell ratio and induction time. Our results indicate that Azk uptake is comparatively efficient in the batch phase. We discovered that the time between Azk uptake and inducing its incorporation into the Fab must be kept short, which suggests that intracellular Azk is consumed and/or degraded. The results obtained in this study are an important step towards the development of efficient production methods for Azk-incorporated proteins in E. coli. The developed process is scalable and provides excellent yields of 2.95 mg functionalized Fab per g CDM, which corresponds to 80% of yield obtained with the wild type Fab. We also identified the cellular uptake of Azk being dependent on the physiological state of the cell as a potential bottleneck in production.

Cell-free bilayer functionalized scaffold for osteochondral tissue engineering.

Osteochondral tissue engineering using layered scaffolds is a promising approach for treating osteochondral defects as an alternative to microfracture procedure, autologous chondrocyte implantation, and cartilage-bone grafting. The team previously investigated the chondrogenesis of mesenchymal stem cells (MSCs) on a polycaprolactone (PCL)/acetylated hyaluronic acid scaffold. The present study first focused on fabricating a novel osteoconductive scaffold utilizing bismuth-nanohydroxyapatite/reduced graphene oxide (Bi-nHAp/rGO) nanocomposite and electrospun PCL. The osteoconductive ability of the scaffold was investigated by evaluating the alkaline phosphatase (ALP) activity and the osteogenic genes expression in the adipose-derived MSCs. The expression of Runx2, collagen I, ALP, and osteocalcin as well as the result of ALP activity indicated the osteoconductive potential of the Bi-nHA-rGO/PCL scaffold. In the next step, a bilayer scaffold containing Bi-nHAp/rGO/PCL as an osteogenic layer and acetylated hyaluronic acid/PCL as a chondrogenic layer was prepared by the electrospinning technique and transplanted into osteochondral defects of rats. The chondrogenic and osteogenic markers corresponding to the surrounding tissues of the transplanted scaffold were surveyed 60 days later by real-time polymerase chain reaction (PCR) and immunohistochemistry methods. The results showed increased chondrogenic (Sox9 and collagen II) and osteogenic (osteocalcin and ALP) gene expression and augmented secretion of collagens II and X after transplantation. The results strongly support the efficacy of this constructed cell-free bilayer scaffold to induce osteochondral defect regeneration.

Antibody fragments functionalized with non-canonical amino acids preserving structure and functionality - A door opener for new biological and therapeutic applications.

Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N6-[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2). Therefore, FTN2Azk variants with different Azk incorporation sites were designed and amber codon suppression was used for production. The functionalized FTN2Azk variants were efficiently produced in fed-batch like µ-bioreactor cultivations in the periplasm of E. coli displaying correct structure and antigen binding affinities comparable to those of wild-type FTN2. Our FTN2Azk variants with reactive handles for diverse conjugates enable tracking of recombinant protein in the production cell, pharmacological studies and translation into new pharmaceutical applications.

A composite bilayer scaffold functionalized for osteochondral tissue regeneration in rat animal model.

Osteochondral defects are defined most typically by damages to both cartilage and subchondral bone tissue. It is challenging to develop bilayered scaffolds that regenerate both of these lineages simultaneously. In the present study, an electrospun bilayer nanofibrous scaffold was designed to repair osteochondral lesions. A nanocomposite of hydroxyapatite, strontium, and reduced graphene oxide were combined with polycaprolactone polymer to fabricate the osteogenic differentiation layer. Additionally, the chondrogenic differentiation layer was also formed using polyethersulfone polymer and benzyl hyaluronan. The physical, mechanical, and chemical properties of the scaffolds were determined, and adipose-derived mesenchymal stem cells were cultured on each layer to evaluate their biocompatibility and differentiation potential. Cell viability, mineralization, alkaline phosphatase enzyme (ALP) expression, and extracellular calcium deposition were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, alizarin red staining, ALP activity, and calcium deposition. Real-time polymerase chain reaction (PCR) was used to assess the expression levels of osteogenic (Collagen I, Runx II, ALP, Osteocalcin) and chondrogenic (Sox9, Collagen II (Col II), Aggrecan) genes. Finally, the osteochondral scaffold was created by electrospinning these two layers for 2 days. The scaffold was grafted into the osteochondral defect of a Wistar rat's knee. 60 days after surgery, real-time PCR, immunohistochemistry (IHC), and hematoxylin and eosin staining were performed. The expression of chondrogenic and osteogenic genes was increased compared to the control group, as confirmed by real-time PCR. Furthermore, IHC revealed a rise in Col II and Collagen X expression. Finally, in vivo and in vitro studies have shown that the electrospun bilayer scaffold is biocompatible, which facilitates osteochondral healing.

Hyperosmolarity benefits cartilage regeneration by enhancing expression of chondrogenic markers and reducing inflammatory markers.

Application of hyperosmolarity can be a promising strategy to promote chondrogenic differentiation in adipose-derived mesenchymal stem cells (ADSCs). Growth factors may promote different signaling pathways in parallel that is why in this study we monitor undesired pathologic or unwanted side effects as well as chondroinductive impacts of hyperosmolarity in differentiating ADSCs. Quantified gene expression, immunocytochemistry, glycosaminoglycan deposition and angiogenic secretion assays performed along with immunoassay. We observed that hyperosmolarity pressure of 480 mOsm over-expressed cartilage specific markers at gene expression level in the extra cellular matrix. Meanwhile, hyperosmolarity of 480 mOsm diminished the expression of cartilage associated pathologic markers, i.e., inflammatory and angiogenic attributes. Certain dose of hyperosmolarity could benefit chondrogenesis in a dual way, first by increasing chondrogenic markers and second by lowering tissue mineralization and angiogenic potential. The chondroprotective potential of hyperosmolarity could have a promising benefit in cartilage cell therapy and tissue engineering.

Acetylated hyaluronic acid effectively enhances chondrogenic differentiation of mesenchymal stem cells seeded on electrospun PCL scaffolds.

Construction of scaffolds which are similar to natural niches regarding both biochemical composition and mechanical characteristics has gained great attention in the field of tissue engineering. However, application of natural polymers, such as hyaluronic acid, is challenging in construction of scaffolds due to physicochemical properties, difficult to use in electrospinning and low cell adhesion rate. In this study, HA was acetylated to make it soluble in high polarity solvent and blended with PCL for construction of nanofibrous composite (ac-HA/PCL) scaffolds. Chondroinductivity of the constructed scaffolds was investigated using human mesenchymal stem cells (hADSCs). The presence of acetyl groups, as well as morphology and biocompatibility of the composite scaffolds were characterized by HNMR, FTIR, SEM and MTT assay respectively. Expression of cartilage-specific genes (SOX9, Col II and Aggrecan) was monitored by Real-time PCR. Significant increase in expression of Sox9 and Col II as the markers of chondrogenic differentiation as well as the results of Alcian blue staining, indicated the chondro-inductive potential of HA/PCL nanofibrous scaffolds. Acetylated HA was biocompatible with chondroinductivity features, therefore it not only had the positive characteristics of natural HA, but also enhanced the cellular attachment and application potential.

Comparative impact of platelet rich plasma and transforming growth factor-ß on chondrogenic differentiation of human adipose derived stem cells.

Introduction: Transforming growth factor-beta (TGF-ß) is known as standard chondrogenic differentiation agent, even though it comes with undesirable side effects such as early hypertrophic maturation, mineralization, and secretion of inflammatory/angiogenic factors. On the other hand, platelet-rich plasma (PRP) is found to have a chondrogenic impact on mesenchymal stem cell proliferation and differentiation, with no considerable side effects. Therefore, we compared chondrogenic impact of TGF-ß and PRP on adipose-derived stem cells (ADSCs), to see if PRP could be introduced as an alternative to TGF-ß. Methods: Differentiation of ADSCs was monitored using a couple of methods including glycosaminoglycan production, miRNAs expression, vascular endothelial growth factor (VEGF)/tumor necrosis factor alpha (TNFα) secretion, alkaline phosphatase (ALP) and calcium content assays. Results: Accordingly, the treatment of differentiating cells with 5% (v/v) PRP resulted in higher glycosaminoglycan production, enhanced SOX9 transcription, and lowered TNFα and VEGF secretion compared to the control and TGF-ß groups. Besides, the application of PRP to the media up-regulated miR-146a and miR-199a in early and late stages of chondrogenesis, respectively. Conclusion: PRP induces in vitro chondrogenesis, as well as TGF-ß with lesser inflammatory and hypertrophic side effects.

Osmolyte Type and the Osmolarity Level Affect Chondrogenesis of Mesenchymal Stem Cells.

The inductive effects of increased osmolarity on chondrogenesis are well approved. However, the effects of the osmolyte agent invoked to induce hyperosmolarity are largely neglected. Herein, we scrutinized how hyperosmotic conditions acquired by addition of different osmolytes would impact chondrogenesis. We briefly assessed whether such conditions would differentially affect hypertrophy and angiogenesis during MSC chondrogenesis. Chondrogenic and hypertrophic marker expression along with VEGF secretion during adipose-derived (AD)-MSC chondrogenesis under three osmolarity levels (350, 450, and 550 mOsm) using three different osmolytes (NaCl, sorbitol, and PEG) were assessed. MTT assay, qRT-PCR, immunocytochemistry, Alcian Blue staining, ELISA, and ALP assays proved osmolyte-type dependent effects of hyperosmolarity on chondrogenesis, hypertrophy, and angiogenesis. At same osmolarity level, PEG had least cytotoxic/cytostatic effect and most prohibitive effects on angiogenesis. As expected, all hyperosmolar conditions led to enhanced chondrogenesis with slightly varying degrees. PEG and sorbitol had higher chondro-promotive and hypertrophy-suppressive effects compared to NaCl, while NaCl had exacerbated hypertrophy. We observed that TonEBP was involved in osmoadaptation of all treatments in varying degrees. Of importance, we highlighted differential effects of hyperosmolarity obtained by different osmolytes on the efficacy of chondrogenesis and more remarkably on the induction/suppression of cartilage pathologic markers. Our study underlies the need for a more vigilant exploitation of physicobiochemical inducers in order to maximize chondrogenesis while restraining unwanted hypertrophy and angiogenesis.

Physical stimulation and scaffold composition efficiently support osteogenic differentiation of mesenchymal stem cells.

BACKGROUND: Despite significant achievements in the field of tissue engineering, simplification and improvement of the existing protocols are of great importance. The use of complex differentiation media, due to the presence of multiple factors, may have some undesired effects on cell health and functions. Thus, minimizing the number of involved factors, while maintaining the differentiation efficiency, provides less costly and controllable conditions. Adipose-derived Mesenchymal stem cells (ASCs), the adult stem cells present in adipose tissue, can be a suitable source of stem cells due to abundant and ease of access. The aim of this study is to optimize the osteogenic differentiation of ASCs by chemical composition of scaffold, in the first step, and then by electromagnetic treatments. METHODS: ASCs were cultured on PVA/PES scaffold and tissue culture polystyrene surfaces (TCPS) and osteogenic differentiation was performed with either osteogenic medium, or electromagnetic field or both. The impact of each treatment on ASCs growth and proliferation was measured by MTT assay. Changes in gene expression levels of osteogenic-specific markers including ALP and RUNX2 were determined by Real Time PCR. Furthermore, alkaline phosphatase activity and calcium deposition were measured. RESULTS: The MTT assay showed the significant effects on cell growth and respiration in scaffold-seeded ASCs treated with electromagnetic field, compared to control TCPS plate. Also, the electromagnetic treatment, increased alkaline phosphatase activity and calcium deposition. Finally, Real Time PCR showed higher expression of ALP and RUNX2 genes in electromagnetic field groups compared to control groups. CONCLUSION: It can be concluded that PVA/PES scaffold used in this study improved the osteogenic capacity of ASCs. Moreover, the osteogenic potential of ASCs seeded on PVA/PES scaffold could be augmented by electromagnetic field without any chemical stimulation.

New Approach for Differentiation of Bone Marrow Mesenchymal Stem Cells Toward Chondrocyte Cells With Overexpression of MicroRNA-140.

Mesenchymal stem cells are widely stimulated by transforming growth factor beta-3 (TGFß3) for chondrocyte differentiation. The objective of our study was to establish a new method for differentiation of human mesenchymal stem cells toward chondrocyte by overexpression of MicroRNA-140 (miR-140), and also this method was compared with method of induction with TGFß3 in high-cell density culture systems. Mesenchymal stem cells were harvested from bone marrow of human. We prepared vectors and then was used for recombinant Lenti virus production in HEK-293 cell. Transducted cells were cultured in monolayer culture system and were harvested after days 7, 14, and 21. Real time polymerase chain reaction (RT-PCR) was performed to evaluate the cartilage-specific genes in the mRNA levels. Also, in order to confirm our results, we have done immunocytochemistry technique. Bone marrow mesenchymal stem cells (BMSCs) were transducted with recombinant Lenti virus, and miR-140 was expressed. Immunocytochemical method confirmed the differentiation of BMSC toward chondrocyte with handling cartilage matrix genes. Also real-time PCR showed that after expression of miR-140 in transducted BMSCs significantly increased gene expression of collagen type II and aggrecan and downregulated expression of collagen type I when compared with the mRNA levels measured in nontransducted BMSCs. These results were compatible compared with TGFß3 induction method as control positive. In this study, we described a new approach and technique that may be applied for differentiation of BMSCs to chondrocyte instead of stimulation with TGFß3. Our data implies that miR-140 is a potent chondrogenic differentiation inducer for BMSCs, and we have shown increasing chondrogenic differentiation by using miR-140 overexpression.

Enhancement of osteogenic differentiation of adipose-derived stem cells by PRP modified nanofibrous scaffold.

Recent developments in bone tissue engineering have paved the way for more efficient and cost-effective strategies. Additionally, utilization of autologous sources has been considered very desirable and is increasingly growing. Recently, activated platelet rich plasma (PRP) has been widely used in the field of bone tissue engineering, since it harbours a huge number of growth factors that can enhance osteogenesis and bone regeneration. In the present study, the osteogenic effects of PRP coated nanofibrous PES/PVA scaffolds on adipose-derived mesenchymal stem cells have been investigated. Common osteogenic markers were assayed by real time PCR. Alkaline phosphate activity, calcium deposition and Alizarin red staining assays were performed as well. The results revealed that the highest osteogenic differentiation occurred when cells were cultured on PRP coated PES/PVA scaffolds. Interestingly, direct application of PRP to culture media had no additive effects on osteogenesis of cells cultured on PRP coated PES/PVA scaffolds or those receiving typical osteogenic factors. The highest osteogenic effects were achieved by the simplest and most cost-effective method, i.e. merely by using PRP coated scaffolds. PRP coated PES/PVA scaffolds can maximally induce osteogenesis with no need for extrinsic factors. The major contribution of this paper to the current researches on bone regeneration is to suggest an easy, cost-effective approach to enhance osteogenesis via PRP coated scaffolds, with no additional external growth factors.

Enhanced Osteogenic Differentiation of Mesenchymal Stem Cells on Electrospun Polyethersulfone/Poly(Vinyl) Alcohol/Platelet Rich Plasma Nanofibrous Scaffolds.

Over the last few decades, great advancements have been achieved in the field of bone tissue engineering (BTE). Containing a great number of growth factors needed in the process of osteogenesis, platelet rich plasma (PRP) has gained a great deal of attention. However, due to the contradictory results achieved in different studies, its effectiveness remains a mystery. Therefore, in this study, we investigated in vitro performance of co-electrospun PRP/poly ether sulfone/poly(vinyl) alcohol (PRP/PES/PVA) composite scaffolds for the osteogenic differentiation of human adipose-derived mesenchymal stem cells. The activated PRP was mixed with PVA solution to be used alongside PES solution for the electrospinning process. Fourier transform infrared spectroscopy, scanning electron microscopy and tensile tests were performed to evaluate the scaffolds. After confirmation of sustained release of protein, osteogenic potential of the co-electrospun PRP/polymer scaffolds was evaluated by measuring relative gene expression, calcium content, and alkaline phosphatase (ALP) activity. Alizarin red and Hematoxylin and Eosin staining were performed as well. The results of ALP activity and calcium content demonstrated the effectiveness of PRP when combined with PRP-incorporated scaffold in comparison with the other tested groups. In addition, the results of tensile mechanical testing indicated that addition of PRP improves the mechanical properties. Taking these results into account, it appears PES/PVA/PRP scaffold treated with PRP 5% enhances osteogenic differentiation most. In conclusion, incorporation of PRP into electrospun PES/PVA scaffold in this study had a positive influence on osteogenic differentiation of AdMSCs, and thus it may have great potential for BTE applications.

The effect of nanofibre-based polyethersulfone (PES) scaffold on the chondrogenesis of human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) have been shown to have promising potential for regenerative medicine and tissue engineering applications. Chondrogenic differentiation of iPSCs is important for application in cartilage tissue engineering. In this study, we considered the effect of nanofibre-based polyethersulfone (PES) scaffold on the chondrogenesis of iPSCs. IPSC cells were cultured on the PES scaffold and scaffold free method. After 21 d, real-time PCR was performed to evaluate the cartilage-specific genes in the mRNA levels. For confirm our results, we have done immunocytochemistry and scanning electron microscopy (SEM) imaging. According to the results, higher significant expressions of common chondrogenic-related genes such as aggrecan, collagen type II and collagen type X were observed in PES seeded human iPSCs when compared to the mRNA levels measured in scaffold free method. Expression of collagen type I down regulated in both methods. Also, both methods were showed a similar pattern of expression of SOX9. Our results showed that nanofibre-based PES scaffold enhanced the chondrogenesis of iPSCs and the highest capacity for differentiation into chondrocyte-like cells. These cells and PES scaffold were demonstrated to have great efficiency for treatment of cartilage damages and lesions.